Preparation Of All-trans Astaxanthin Stereoisomers And Inhibition Study Of α-Glucosidase And Aldose Reductase

Type 2 diabetes and its complications are one of the important health issues of global concern.In order to reduce the incidence of postprandial hyperglycemia and chronic hyperglycemiain the course of its onset,it is an effective method to inhibit key enzymes.Chemically synthesized enzyme inhibitors have side effects in the treatment of hyperglycemia,so the search for functional compounds that modulate hyperglycemia from natural sources has aroused widespread interest.Naturally derived all-trans astaxanthin has antioxidant and hypoglycemic effects,but the inhibitory effect on diabetes-related enzymes is unknown.In the study,all-trans astaxanthin stereoisomers were purified from Phaffia rhodozyma and Haematococcus pluvialis used an optimized method.Based on enzy me kinetics,circular dichroism.fluorescence spectrum and molecular docking et al.methods,the inhibition mechanisms of all-trans astaxanthins stereoisomers on α-glucosidase and aldose reductase were further studied.The main results are as follows:The conditions of semi-preparative HPLCwere as follow:Methanol-Methyl tert-butyl ether(90%:10%,v/v),chromatographic column was a CHIRALPAK? IC 83325 chiral preparative column(10 mm × 250 mm,5 μm),maximum flow rate was 3.06 mL/min.the maximum injection volume was 0.32 mg.the column temperature was 35℃,and the detection wavelength was 474 nm.Using this method and identified by HPLC-UV.HPLC-ESI-MS and 1H NMR,3R,3’R astaxanthin(R AST.the purity 97.5%)was isolated from P.rhodozyma and 3S.3’ S astaxanthin(S AST.the purity 97.9%)was isolated from H.pluvialis.The inhibitory effect of R AST on α-glucosidase was the strongest among astaxanthin stereoisomers.The IC50 value of R AST was 28.87±0.18μmol/L,and the inhibition type was competitive inhibition.When R AST interacted with α-glucosidase,the content of α-helix decreased,the content of β-sheet increased.Increasing the concentration of R AST could reduce the fluorescence intensity at the maximum wavelength,which indicated that R AST quenched the fluorescence of α-glucosidase.Computer simulation and docking analysis revealed that R AST had the lowest binding free energy and stably occupied catalytic center,resulting in the inaccessibility of the substrate,and inhibited enzymatic activity.The inhibitory effect of R AST on aldose reductase was the strongest among astaxanthin stereoisomers.The IC50 value of R AST was 13.19±0.36 μmol/L,and the inhibition type was competitive inhibition.When R AST interacted with aldose reductase,the contents of α-helix andβ-turn decreased,the content of β-sheet increased,and the secondary structure changed.Increasing the concentration of R AST could reduce the fluorescence intensity at the maximum wavelength,which indicated that R AST quenched the fluorescence of aldose reductase and changedits tertiary structure.The result of computer simulation and molecular docking showed that the combination of R AST and the enzyme caused the position of key amino acid residues in the hydrophobic pocket to change,resulting in changing the overall structure of the enzyme and reducing enzymatic activity.The results of this study provide theoretical basis and data support for the separation and preparation of all-trans astaxanthin stereoisomers and the inhibition mechanisms of enzymes.At the same time,it also provides a scientific basis for the development of highly effective natural antidiabetic drugs using astaxanthin.